Nitrous oxide (N₂O) is a powerful greenhouse gas that contributes to climate change. Denitrification is one of the largest sources of N₂O in soils. The soybean endosymbiont Bradyrhizobium diazoefficiens is a model for rhizobial denitrification studies since, in addition to fixing N₂, it has the ability to grow anaerobically under free-living conditions by reducing nitrate from the medium through the complete denitrification pathway. This bacterium contains a periplasmic nitrate reductase (Nap), a copper (Cu)-containing nitrite reductase (NirK), a c-type nitric oxide reductase (cNor), and a Cu-dependent nitrous oxide reductase (Nos) encoded by the napEDABC, nirK, norCBQD and nosRZDFYLX genes, respectively. In this work, an integrated study of the role of Cu in B. diazoefficiens denitrification has been performed. A notable reduction in nirK, nor, and nos gene expression observed under Cu limitation was correlated with a significant decrease in NirK, NorC and NosZ protein levels and activities. Meanwhile, nap expression was not affected by Cu, but a remarkable depletion in Nap activity was found, presumably due to an inhibitory effect of nitrite accumulated under Cu-limiting conditions. Interestingly, a post-transcriptional regulation by increasing Nap and NirK activities, as well as NorC and NosZ protein levels, was observed in response to high Cu. Our results demonstrate, for the first time, the role of Cu in transcriptional and post-transcriptional control of B. diazoefficiens denitrification. Thus, this study will contribute by proposing useful strategies for reducing N₂O emissions from agricultural soils.

Summary: The FixK ₂ protein plays a pivotal role in a complex regulatory network, which controls genes for microoxic, denitrifying, and symbiotic nitrogen‐fixing lifestyles in Bradyrhizobium diazoefficiens . Among the microoxic‐responsive FixK ₂ ‐activated genes are the fixNOQP operon, indispensable for respiration in symbiosis, and the nnrR regulatory gene needed for the nitric‐oxide dependent induction of the norCBQD genes encoding the denitrifying nitric oxide reductase. FixK ₂ is a CRP/FNR‐type transcription factor, which recognizes a 14 bp‐palindrome (FixK ₂ box) at the regulated promoters through three residues (L195, E196, and R200) within a C‐terminal helix‐turn‐helix motif. Here, we mapped the determinants for discriminatory FixK ₂ ‐mediated regulation. While R200 was essential for DNA binding and activity of FixK ₂ , L195 was involved in protein–DNA complex stability. Mutation at positions 1, 3, or 11 in the genuine FixK ₂ box at the fixNOQP promoter impaired transcription activation by FixK ₂ , which was residual when a second mutation affecting the box palindromy was introduced. The substitution of nucleotide 11 within the NnrR box at the norCBQD promoter allowed FixK ₂ ‐mediated activation in response to microoxia. Thus, position 11 within the FixK ₂ /NnrR boxes constitutes a key element that changes FixK ₂ targets specificity, and consequently, it might modulate B. diazoefficiens lifestyle as nitrogen fixer or as denitrifier.

The soybean endosymbiont Bradyrhizobium diazoefficiens harbours the complete denitrification pathway that is catalysed by a periplasmic nitrate reductase (Nap), a copper (Cu)-containing nitrite reductase (NirK), a c-type nitric oxide reductase (cNor), and a nitrous oxide reductase (Nos), encoded by the napEDABC, nirK, norCBQD, and nosRZDFYLX genes, respectively. Induction of denitrification genes requires low oxygen and nitric oxide, both signals integrated into a complex regulatory network comprised by two interconnected cascades, FixLJ–FixK₂–NnrR and RegSR–NifA. Copper is a cofactor of NirK and Nos, but it has also a role in denitrification gene expression and protein synthesis. In fact, Cu limitation triggers a substantial down-regulation of nirK, norCBQD, and nosRZDFYLX gene expression under denitrifying conditions. Bradyrhizobium diazoefficiens genome possesses a gene predicted to encode a Cu-responsive repressor of the CsoR family, which is located adjacent to copA, a gene encoding a putative Cu⁺-ATPase transporter. To investigate the role of CsoR in the control of denitrification gene expression in response to Cu, a csoR deletion mutant was constructed in this work. Mutation of csoR did not affect the capacity of B. diazoefficiens to grow under denitrifying conditions. However, by using qRT-PCR analyses, we showed that nirK and norCBQD expression was much lower in the csoR mutant compared to wild-type levels under Cu-limiting denitrifying conditions. On the contrary, copA expression was significantly increased in the csoR mutant. The results obtained suggest that CsoR acts as a repressor of copA. Under Cu limitation, CsoR has also an indirect role in the expression of nirK and norCBQD genes.

FixK₂ is a CRP/FNR-type transcription factor that plays a central role in a sophisticated regulatory network for the anoxic, microoxic and symbiotic lifestyles of the soybean endosymbiont Bradyrhizobium diazoefficiens. Aside from the balanced expression of the fixK₂ gene under microoxic conditions (induced by the two-component regulatory system FixLJ and negatively auto-repressed), FixK₂ activity is posttranslationally controlled by proteolysis, and by the oxidation of a singular cysteine residue (C183) near its DNA-binding domain. To simulate the permanent oxidation of FixK₂, we replaced C183 for aspartic acid. Purified C183D FixK₂ protein showed both low DNA binding and in vitro transcriptional activation from the promoter of the fixNOQP operon, required for respiration under symbiosis. However, in a B. diazoefficiens strain coding for C183D FixK₂, expression of a fixNOQP’-‘lacZ fusion was similar to that in the wild type, when both strains were grown microoxically. The C183D FixK₂ encoding strain also showed a wild-type phenotype in symbiosis with soybeans, and increased fixK₂ gene expression levels and FixK₂ protein abundance in cells. These two latter observations, together with the global transcriptional profile of the microoxically cultured C183D FixK₂ encoding strain, suggest the existence of a finely tuned regulatory strategy to counterbalance the oxidation-mediated inactivation of FixK₂ in vivo.

Soybean (Glycine max.) is one of the most important legumes cultivated worldwide. Its productivity can be altered by some biotic and abiotic stresses like global warming, soil metal pollution or over-application of herbicides like paraquat (1,1’-dimethyl-4,4’-bipyridinium dichloride). In this study, the effect of oxidative stress produced by paraquat addition (0, 20, 50 and 100 µM) during plant growth on symbiotic nitrogen fixation (SNF) and functionality of Bradyrhizobium diazoefficiens-elicited soybean nodules were evaluated. Results showed that the 50 µM was the threshold that B. diazoefficiens can tolerate under free-living conditions. In symbiosis with soybean, the paraquat addition statistically reduced the shoot and root dry weight of soybean plants, and number and development of the nodules. SNF was negatively affected by paraquat, which reduced total nitrogen content and fixed nitrogen close to 50% when 100 µM was added. These effects were due to the impairment of nodule functionality and the increased oxidative status of the nodules, as revealed by the lower leghaemoglobin content and the higher lipid peroxidation in soybean nodules from paraquat-treated plants.

Bradyrhizobium diazoefficiens can live inside soybean root nodules and in free-living conditions. In both states, when oxygen levels decrease, cells adjust their protein pools by gene transcription modulation. PhaR is a transcription factor involved in polyhydroxyalkanoate (PHA) metabolism but also plays a role in the microaerobic network of this bacterium. To deeply uncover the function of PhaR, we applied a multipronged approach, including the expression profile of a phaR mutant at the transcriptional and protein levels under microaerobic conditions, and the identification of direct targets and of proteins associated with PHA granules. Our results confirmed a pleiotropic function of PhaR, affecting several phenotypes, in addition to PHA cycle control. These include growth deficiency, regulation of carbon and nitrogen allocation, and bacterial motility. Interestingly, PhaR may also modulate the microoxic-responsive regulatory network by activating the expression of fixK₂ and repressing nifA, both encoding two transcription factors relevant for microaerobic regulation. At the molecular level, two PhaR-binding motifs were predicted and direct control mediated by PhaR determined by protein-interaction assays revealed seven new direct targets for PhaR. Finally, among the proteins associated with PHA granules, we found PhaR, phasins, and other proteins, confirming a dual function of PhaR in microoxia.